The invention relates to nematode-inducible regulatory DNA sequences which can be used for expressing DNA sequences in plant cells. The invention further comprises chimeric DNA comprising said regulatory DNA sequences operably linked to DNA to be expressed in plant cells, as well as plants containing such chimeric DNA in their cells. The invention further relates to methods for making plants that are resistant, or at least less susceptible to plant parasitic nematodes, or their effects, as well as to cells, plants and parts thereof.
In International patent application WO92/17054, a method is disclosed for the identification and subsequent isolation of nematode responsive regulatory DNA sequences from Arabidopsis thaliana. 
In WO 92/21757 several regulatory DNA sequences have been isolated from Lycopersicon esculentum, which are responsive to the root-knot nematode Meloidogyne incognita. Some of these regulatory sequences (LEMMI""s, for Lycopersicon esculentumxe2x80x94Meloidogyne incognita) are stimulated, whereas others appear to be repressed by the nematode. It is not known whether any of the inducible regulatory sequences are stimulated by a broader range of nematodes.
Another regulatory sequence that is inducible by the root-knot nematode Meloidogyne incognita is disclosed in WO 93/06710. A disadvantage of this regulatory sequence TobRb7 is that it is not activated by a number of cyst nematodes, among which the Heterodera and Globoelera species. This makes the TobRB7 sequence unsuitable for use in chimeric constructs aiming at, for example, cyst nematode resistance in potato.
It is an object of the invention to provide regulatory DNA sequences which are inducible by both cyst and root knot nematodes and which can be used to express heterologous DNA sequences under their control inside the feeding structure of the nematode, preferably, but not necessarily in a substantially feeding site specific way.
The invention provides a DNA fragment obtainable from Arabidopsis thaliana that is capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant. Preferred according to the invention are sequences represented by nucleotides 1 to 3484 in SEQIDNO: 1. Also envisaged are portions or variants of a DNA fragment according to the invention capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant. A still further preferred aspect of the invention comprises a regulatory DNA fragment that is substantially nematode feeding site-specific.
Further embodiments of the invention comprise chimeric DNA sequences comprising in the direction of transcription a regulatory DNA fragment according to the invention and a DNA sequence to be expressed under the transcriptional control thereof and which is not naturally under transcriptional control of said DNA fragment. Preferred among the chimeric DNA sequences according to the invention are those wherein the DNA sequence to be expressed causes the production of a plant cell-disruptive substance, such as barnase. In a different embodiment the cell-disruptive substance comprises RNA complementary to RNA essential to cell viability. Yet in another embodiment the DNA sequence to be expressed causes the production of a substance toxic to the inducing nematode.
The invention finds further use in a replicon comprising a DNA fragment or chimeric DNA sequence according to the invention, a microorganism containing such a replicon, as well as plant cells having incorporated into their genome a chimeric DNA sequence according to the invention. Further useful embodiments are a root system of a plant essentially consisting of cells according to the invention, as well as full grown plants essentially consisting of cells according to the invention, preferably a dicotyledonous plant, more preferably a potato plant. Also envisaged are plants grafted on a root system according to the invention, as well as plant parts selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood and crops comprising such plants.
The invention also encompasses the use of a DNA fragment according to the invention for identifying subfragments capable of promoting transcription of an associated DNA sequence in a plant. Also envisaged is the use of a chimeric DNA sequence according to the invention for transforming plants. The invention further provides the use of a fragment, portion or variant of a regulatory DNA according to the invention for making hybrid regulatory DNA sequences.
The following figures further illustrate the invention.